Abstract Applications of customizable nucleases such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9) have enabled efficient and precise gene correction in vitro, and hold promises for eventually achieving in vivo gene correction/therapy. However, to apply CRISPR/Cas9 in therapeutic settings, several major challenges remain to be addressed: (i) homologous recombination (HR), even with the help of Cas9, is still of low efficiency; (ii) Cas9 is associated with off-target effects; and (iii) there is a lack of an efficient virus-free system to deliver CRISPR/Cas9 elements in vivo. The present proposal focuses on the challenge of lack of an efficient non-viral in vivo delivery system. We recently developed novel hyperbranched polymers (HPs) with high nucleic acid binding affinity, negligible cytotoxicity, and achieved satisfactory delivery of microRNAs both in vitro and in vivo. Here we propose to develop hyperbranched HP- based system for effective delivery of Cas9 plasmid DNA (pDNA). In Phase I, we will work to formulate and test new HPs for effectively packaging of Cas9 pDNA, and evaluate the safety and efficacy of these HPs in vitro. In Phase II, we will expand the capability of HPs for Cas9 in vivo, and apply HPs for Cas9 therapeutics in animal models. Success of the proposed work will have significant impacts on basic and translational research and accelerate the development of Cas9 therapeutics.